380 FATTY SUBSTANCES (GENERAL METHODS) acid, the number of c.c. of thiosulphate used corresponds with 0-2 gram of iodine. 5. Chloroform, which should be pure. PROCEDURE. In a thin glass bulb the fatty substance (as it is, if liquid, or fused if a solid, but always previously dehydrated and filtered : see i ; Preparation of the Sample) is weighed, 0-1-0-2 gram being taken of a drying- oil, 0-2-0-3 gram of a semi-drying oil, 0-3-0-4 gram of a non-drying oil or 0-4-0-8 gram of a solid fat. The bulb is placed in a half-litre glass bottle with a tight ground stopper, the bottle being held obliquely and suddenly shaken so as to break the bulb against the walls. The fat is then dissolved in 15 c.c. of chloroform and treated with 25 c.c. of the mixture in equal volumes of the iodine and mercuric chloride solutions (prepared about 48 hours earlier), care being taken in all cases to empty the pipette in the same way so that exactly the same volume of solution is used. The liquid is carefully shaken and the bottle stoppered and kept in a dark cool place (15-18°) for 4-6 hours with non-drying or semi-drying oils or for 18-24 hours with drying oils. At the end of this time, 15-20 c.c. of the potassium iodide solution are introduced, the stopper, neck and walls of the bottle being washed with this solution and with about 200 c.c. of distilled water subsequently added. The excess of iodine is then titrated with the thio- sulphate solution, which is slowly run in until the aqueous liquid and the chloroform beneath appear only pale yellow; about 5 c.c. of the starch solution are then added and the titration completed. This test is always made in duplicate and at the same time two blank experiments are carried out with the same proportions of solutions and tinder the same conditions, but without the fatty substance. The amounts of iodine in the two checks are titrated one before and the other after the actual test, the mean value being taken. This mean is deducted from the mean value obtained in the test? with the fat, the remainder representing the amount of iodine absorbed by the fat, and this, calculated as percentage, is the iodine number. B. Wijs's Method.—In this method, the alcoholic solution of iodine and mercuric chloride is replaced by an acetic acid solution of iodine mono- chloride prepared as follows : 8 grams of pure iodine trichloride and 8-5 grams of iodine are dis- solved separately in pure glacial acetic acid (99%) on a water-bath and in dry, closed vessels to avoid absorption of moisture. When cool, the two solutions are transferred to the same i-litre flask and made up to volume with pure glacial acetic acid. It is necessary to ascertain that the acetic acid is at least 99% and that it is pure : when heated with potassium dichromate and cone, sulphuric acid ic should give no coloration even after some time. Further, to dissolve the fatty substance use is made of pure carbon tetmchloride (also to be tested with dichromate and sulphuric acid, as with the acetic acid). The solutions 4, 2 and 3 (sodium thiosulphate, potassium iodide and starch) of Hubl's method are used also in this case. The procedure is the same as with Hubl's method, excepting that the1, this giving the acetyl saponification number.